E. coli O157:H7 is an emerging pathogen of major importance. Disease caused by E. coli O157:H7 is characterized by diarrhea, hemorrhagic colitis, and the potentially fatal complication, hemolytic uremic syndrome (HUS). Shiga toxin (Stx) is a major virulence factor of E. coli O157:H7. Two major antigenic variants of Shiga toxin, Stx1 and Stx2, share 55% amino acid homology, however Stx2 production has been associated with progression to severe disease, including HUS. We propose to examine two aspects of Shiga toxin in the pathogenesis of disease caused by E. coli O157:H7 - regulation of Shiga toxin production in the intestine and the molecular basis for the difference in potency between Stx1 and Stx2. The genes for Shiga toxin are encoded on bacterial viruses. Regulation of toxin production is strongly linked to factors that affect the virus life cycle, specifically factors that induce the viral lytic cycle, for example antibiotics such as ciprofloxicin. In Specific Aim 1 we will characterize factors that influence Stx2 expression in vitro and in vivo. In addition to amount of toxin production, the type of Shiga toxin produced during infection can influence the severity of disease, with Stx2 being by far the more potent toxin. While it is clear that Stx2 can induce cellular death, it is not clear that cellular death is required for development of HUS, and toxicity may occur at the organismal level. In Specific Aim 2 we will characterize hybrid Stx2/Stx1 mutants to determine why Stx2 is more potent than Stx1 using in vitro cell culture models. In Specific Aim 3 we will determine the potency of the hybrid mutants in vivo using a mouse model of disease. Understanding the viral, bacterial and host factors that influence Shiga toxin production and potency could lead to treatments that reduce the incidence of fatal disease.